Glucoamylase variants and polynucleotides encoding same

Abstract

The present invention relates to glucoamylase variants having improved thermostability. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

The invention claimed is: 
     
       1. An isolated polynucleotide encoding a polypeptide, wherein the polypeptide is a glucoamylase variant comprising a substitution or deletion at one or more positions corresponding to positions 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, 26, 31, 33, 34, 65, 72, 74, 79, 80, 103, 105, 112, 161, 172, 218, 220, 221, 245, 253, 255, 279, 325, 327, 359, 364, 370, 375, 377, 405, 445, 447, 460, 463, 465, 468, 477, 501, 502, 504, 516, 524, 526, 563, 564, 568, 571 of the polypeptide of SEQ ID NO: 3, wherein the variant has glucoamylase activity and at least 70 percent sequence identity to the polypeptide of SEQ ID NO: 3. 
     
     
       2. A nucleic acid construct or expression vector comprising the polynucleotide of  claim 1  operably linked to one or more control sequences that direct the production of the polypeptide in an expression host. 
     
     
       3. A recombinant host cell comprising the polynucleotide of  claim 1  operably linked to one or more control sequences that direct the production of the polypeptide. 
     
     
       4. A method of producing a polypeptide, comprising:
 (a) cultivating the host cell of  claim 3  under conditions conducive for production of the polypeptide; and 
 (b) recovering the polypeptide. 
 
     
     
       5. The polynucleotide of  claim 1 , wherein the variant comprises a substitution or deletion in at least one position selected from position 65, 327, 501, 504 of SEQ ID NO: 3. 
     
     
       6. The polynucleotide of  claim 1 , wherein the variant comprises or consists of one or more substitutions selected from the group consisting of T65A, Q327F, E501V, Y504T, Y504*. 
     
     
       7. The polynucleotide of  claim 1 , wherein the variant comprises at least one of the following substitutions or combinations of substitutions:
 T65A; or 
 Q327F; or 
 E501V; or 
 Y504T; or 
 Y504*; or 
 T65A+Q327F; or 
 T65A+E501V; or 
 T65A+Y504T; or 
 T65A+Y504*; or 
 Q327F+E501V; or 
 Q327F+Y504T; or 
 Q327F+Y504*; or 
 E501V+Y504T; or 
 E501V+Y504*; or 
 T65A+Q327F+E501V; or 
 T65A+Q327F+Y504T; or 
 T65A+E501V+Y504T; or 
 Q327F+E501V+Y504T; or 
 T65A+Q327F+Y504*; or 
 T65A+E501V+Y504*; or 
 Q327F+E501V+Y504*; or 
 T65A+Q327F+E501V+Y504T; or 
 T65A+Q327F+E501V+Y504*. 
 
     
     
       8. The polynucleotide of  claim 1 , wherein the variant comprises at least one of the following combinations of substitutions:
 E501V+Y504T; 
 T65A+K161S; 
 T65A+Q405T; 
 T65A+Q327W; 
 T65A+Q327F; 
 T65A+Q327Y; 
 P11F+T65A+Q327F; 
 R1K+D3W+K5Q+G7V+N8S+T10D+P11S+T65A+Q327F; 
 P2N+P4S+P11F+T65A+Q327F; 
 P11F+D26C+K330+T65A+Q327F; 
 P2N+P4S+P11F+T65A+Q327W+E501V+Y504T; 
 R1E+D3N+P4G+G6R+G7A+N8A+T10D+P11D+T65A+Q327F; 
 P11F+T65A+Q327W; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; 
 P11F+T65A+Q327W+E501V+Y504T; 
 T65A+Q327F+E501V+Y504T; 
 T65A+S105P+Q327W; 
 T65A+S105P+Q327F; 
 T65A+Q327W+S364P; 
 T65A+Q327F+S364P; 
 T65A+S103N+Q327F; 
 P2N+P4S+P11F+K34Y+T65A+Q327F; 
 P2N+P4S+P11F+T65A+Q327F+D445N+V447S; 
 P2N+P4S+P11F+T65A+I172V+Q327F; 
 P2N+P4S+P11F+T65A+Q327F+N502*; 
 P2N+P4S+P11F+T65A+Q327F+N502T+P563S+K571E; 
 P2N+P4S+P11F+R31S+K33V+T65A+Q327F+N564D+K571S; 
 P2N+P4S+P11F+T65A+Q327F+S377T; 
 P2N+P4S+P11F+T65A+V325T+Q327W; 
 P2N+P4S+P11F+T65A+Q327F+D445N+V447S+E501V+Y504T; 
 P2N+P4S+P11F+T65A+I172V+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+S377T+E501V+Y504T; 
 P2N+P4S+P11F+D26N+K34Y+T65A+Q327F; 
 P2N+P4S+P11F+T65A+Q327F+I375A+E501V+Y504T; 
 P2N+P4S+P11F+T65A+K218A+K221D+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; 
 P2N+P4S+T10D+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+F12Y+T65A+Q327F+E501V+Y504T; 
 K5A+P11F+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+T10E+E18N+T65A+Q327F+E501V+Y504T; 
 P2N+T10E+E18N+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T568N; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+K524T+G526A; 
 P2N+P4S+P11F+K34Y+T65A+Q327F+D445N+V447S+E501V+Y504T; 
 P2N+P4S+P11F+R31S+K33V+T65A+Q327F+D445N+V447S+E501V+Y504T; 
 P2N+P4S+P11F+D26N+K34Y+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+F80*+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+K112S+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; 
 P2N+P4S+P11F+T65A+Q327F+E501V+N502T+Y504*; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+S103N+Q327F+E501V+Y504T; 
 K5A+P11F+T65A+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+E501V+Y504T+T516P+K524T+G526A; 
 P2N+P4S+P11F+T65A+V79A+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+V79G+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+V79I+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+V79L+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+V79S+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+L72V+Q327F+E501V+Y504T; 
 S255N+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+E74N+V79K+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+G220N+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Y245N+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q253N+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+D279N+Q327F+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+S359N+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+D370N+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+V460S+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+V460T+P468T+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+T463N+E501V+Y504T; 
 P2N+P4S+P11F+T65A+Q327F+S465N+E501V+Y504T; or 
 P2N+P4S+P11F+T65A+Q327F+T477N+E501V+Y504T. 
 
     
     
       9. The polynucleotide of  claim 1 , wherein the variant has an improved property relative to a parent, wherein the improved property is reduced sensitivity to protease degradation. 
     
     
       10. The polynucleotide of  claim 1 , wherein the variant has an improved property relative to the parent, wherein the improved property is improved thermostability. 
     
     
       11. The host cell of  claim 3 , wherein the host cell is a fungal host cell. 
     
     
       12. The host cell of  claim 11 , wherein the fungal host cell is a yeast host cell. 
     
     
       13. The host cell of  claim 12 , wherein the yeast host cell is a  Candida, Hansenula, Kluyveromyces, Saccharomyces, Saccharomyces, or Yarrowia  cell. 
     
     
       14. The host cell of  claim 12 , wherein the yeast host cell is a  Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis , or  Yarrowia lipolytica  cell. 
     
     
       15. The host cell of  claim 11 , wherein the fungal host cell is a filamentous fungal host cell. 
     
     
       16. The host cell of  claim 15 , wherein the filamentous fungal host cell is an  Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , or  Trichoderma  cell. 
     
     
       17. The host cell of  claim 15 , wherein the filamentous fungal host cell is an  Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei , or  Trichoderma viride  cell.